The isolation of human fibrinogen has traditionally been carried out by classical plasma fractionation methods. Fibrinogen is precipitated from plasma either with ethanol (Blomback and Blomback, 1956), ammonium sulphate (Takeda, 1996), β alanine/glycine (Jakobsen and Kieruif, 1976), polymers (polyethelene glycol) and low ionic strength solutions (Holm, 1985) with relative high yield and homogeneity.
Further purification of fibrinogen precipitates can be achieved by ion-exchange chromatography conditions (Stathakis et al, 1978) and affinity chromatography (Kuyas et al, 1990). Specific contaminants can be absorbed out for example fibronectin on immobilised gelatine and plasminogen an immobilised lysine (Vuento et al, 1979).
Precipitation methods are widely used for the manufacture of commercial fibrinogen. Chromatographic methods are now being explored as an alternative or to improve the purity of fibrinogen concentrates.
WO 99/37680 describes a method for the large scale separation of fibrinogen from other blood proteins in human blood plasma. The process involves the use of a heparin precipitated paste as a starting material for the purification of fibrinogen. The heparin precipitated paste is a by-product from the manufacturing process of Factor VIII (Antihaemophilic Factor, AHF).
Attempts to produce fibrinogen free of plasminogen or to purify plasminogen itself have been widely published in the literature. The most common method is to utilise the ability of lysine to bind to one of the two “kringles” in the plasminogen molecule. The use of affinity chromatography step was first disclosed in a paper published by Deutsch and Mertz in 1970. Baxter International Inc. utilised this technology, which incorporated the use of lysine-sepharose material in a dedicated step to remove plasminogen from their fibrinogen as disclosed in the patent WO 95/25748 for the large scale manufacture of a fibrinogen concentrate free of destabilising levels of plasminogen product. Other techniques published in the scientific literature again utilise the binding of either lysine or ε-amino caproic acid. However, they are employed to alter the solubility of the plasminogen molecule. Following the addition of lysine to a dilute fibrinogen solution, the subsequent solution is then precipitated in the presence of 7% ethanol. Removal of plasminogen is stated at greater than 90% with a repeat of the step leading to total removal of the contaminant (Mosesson, 1962). Precipitation methods are widely used for the manufacture of commercial fibrinogen, however, the work published by Mosesson (1962) relys on a dilute solution of fibrinogen which is not a practical process for implementation at a production scale.
The use of ion-exchange chromatography and ε-amino caproic acid to bind and elute plasminogen independent of pH or ionic strength was disclosed in a patent (WO 94/00483) lodged in 1994 by Novo Nordisk A/S describing the purification of kringle containing proteins. This method chooses S-sepharose as the resin of choice. Also, a combination of gel filtration and ion-exchange chromatography has been utilised to purify plasminogen. (Robbins et al, 1965).